Freshly frozen tissues (≤30 mm3) were homogenized using the MagNA Lyser Instrument (Roche, Basel, Switzerland; 03358968001) and MagNA Lyser Green Beads (Roche; 03358941001). Total RNA from freshly frozen tissues and endometrioid adenocarcinoma cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany; 74104), and complementary DNA was synthesized from genomic DNA-purified RNAs using qPCR-RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan; FSQ-301). The mRNA expression was measured using KOD SYBR qPCR Mix (TaKaRa Bio, Shiga, Japan; QKD-201X5) and QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific; A40425). Relative gene expression was analyzed using the 2−ΔΔCt method. The primer sequences for RT-qPCR are listed in Table S2 . The experiment was performed in triplicate [24 (link)].
Qpcr rt master mix with gdna remover
Manufactured by Toyobo
Sourced in Japan
QPCR RT Master Mix with gDNA Remover is a laboratory reagent designed for real-time quantitative reverse transcription PCR (RT-qPCR) analysis. The product includes a reverse transcriptase enzyme and a DNA removal agent to eliminate genomic DNA contamination, enabling accurate gene expression quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm ll, by Takara Bio (2 mentions)
Magna lyser green beads, by Roche (1 mentions)
Kod sybr qpcr mix, by Takara Bio (1 mentions)
Quantstudio 1 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Magna lyser instrument, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Truseq stranded mrna lt sample prep kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Sybr green realtime master mix, by Toyobo (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Lightcycler 480 real time pcr instrument, by Roche (1 mentions)
Thermal cycler dice real time system, by Takara Bio (1 mentions)
8 protocols using qpcr rt master mix with gdna remover
1
RNA Extraction and qPCR Quantification
2
Quantitative RT-PCR Analysis of Bovine Mammary Epithelial Cells
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Total RNA was isolated from bovine MECs using TRIzol (Invitrogen, USA), according to the manufacturer’s protocol. For quantitative real-time (qRT) polymerase chain reaction (PCR), cDNA was synthesized from total RNA using a qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). The qRT-PCR used a SYBR Premix Ex Taq II (TaKaRa Biotechnology, Kusatsu, Japan). Primers used for quantitative PCR are shown in Table 1 . Relative gene expression was quantified using the 2−ΔΔCT comparative method and is represented as values relative to control. β-Actin (ACTB) was used as the reference gene. The sensitivity of reactions and amplification of contaminating products, such as extensions of self-annealed primers, were evaluated by amplifying serial cDNA dilutions. The specificity of amplified PCR products is analysed by the dissociation curve and agarose gel electrophoresis. Data analyses followed the manufacturer’s instructions.
3
Transcriptome Sequencing of Cell Samples
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The total RNA of cells was isolated and reverse transcribed by qPCR RT Master Mix with gDNA Remover (Toyobo, #FSQ-301). RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies). Then the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, #20020594) according to the manufacturer's instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd (Shanghai, China) and Lianchuan Biotech Co., Ltd (Hangzhou, China). The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150 bp paired-end reads were generated. Raw data (raw reads) of fastq format were firstly processed using Trimmomatic to remove low-quality reads and obtain the clean reads. Then about 48.3 M clean reads for each sample were retained for subsequent analyses. The clean reads were mapped to the human genome (GRCh38) using HISAT2. FPKM of each gene was calculated using Cufflinks, and HTSeq-count obtained the read counts of each gene. Differential expression analysis was performed using the DESeq (2012) R package. P-value <0.05 and fold change >2 or fold change <0.5 was regarded as significantly differential expression. Hierarchical cluster analysis of differentially expressed genes (DEGs) was performed to demonstrate the expression pattern of genes in different groups and samples.
4
Quantitative gene expression analysis in hypothalamus
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The hypothalamus tissues or cells were collected, and total RNA was extracted with TRIzol reagent (Invitrogen, 15596026) following the manufacturer’s instructions. 500 ng of total RNA from each sample was reverse transcribed to cDNA with qPCR RT Master Mix with gDNA Remover (Toyobo, FSQ-301). According to the manufacturer’s instructions, qPCR was performed using SYBR Green Realtime Master Mix (Toyobo, QPK-201). Each quantitative reaction was performed in triplicate. To normalize the expression data, 18 s was used as the internal control gene. Relative gene expression levels were calculated using the 2-ΔΔCt method. The primer sequences used for the PCR amplification are presented in Table 3 .
Primer sequences for qPCR.
| Primers | Sequences | |
|---|---|---|
| Tnfa | Forward | 5’-AGTCCGGGCAGGTCTACTTT-3’ |
| Reverse | 5’-GGTCACTGTCCCAGCATCTT-3’ | |
| Il-1b | Forward | 5’-CCCAACTGGTACATCAGCAC-3’ |
| Reverse | 5’-TCTGCTCATTCACGAAAAGG-3’ | |
| Ccl2 | Forward | 5’-TGAATGTGAAGTTGACCCGT-3’ |
| Reverse | 5’-AAGGCATCACAGTCCGAGTC-3’ | |
| Nfkb1 | Forward | 5’-ATGGCAGACGATGATCCCTAC-3’ |
| Reverse | 5’-TGTTGACAGTGGTATTTCTGGTG-3’ | |
| Ripk1 | Forward | 5’-GACAGACCTAGACAGCGGAG-3’ |
| Reverse | 5’-CCAGTAGCTTCACCACTCGAC-3’ | |
| Ripk3 | Forward | 5’-GGTGGTGCTACCAAGGAGTT-3’ |
| Reverse | 5’-GAGATGGAAGACACGGCACT-3’ | |
| Mlkl | Forward | 5’-TTAGGCCAGCTCATCTATGAACA-3’ |
| Reverse | 5’-TGCACACGGTTTCCTAGACG-3’ | |
| 18 s | Forward | 5’-GACTCAACACGGGAAACCTC-3’ |
| Reverse | 5’-TAACCAGACAAATCGCTCCAC-3’ |
5
Quantitative Real-Time PCR Protocol
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Total RNA from cells and tissues was extracted using SteadyPure Universal RNA Extraction Ki according to the manufacturer’s instructions. (AG21017, China). Reverse transcription was performed using the qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was carried out on a LightCycler 480 Real-Time PCR instrument (Roche, Basel, Switzerland) using SYBR Green Real-time PCR Master Mix (Toyobo). Analysis was performed using the 2−∆∆Ct method, with GAPDH as the endogenous control. All primer pairs were purchased from Tsingke (Beijing, China), and all sequences are provided in Additional file 1 : Table S1.
6
Quantification of mRNA Expression
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Total RNA was extracted from the HC11 cells using TRIzol reagent (Life Technologies) following the manufacturer’s protocol. A qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) was utilized for reverse transcription. Quantitative real-time PCR was carried out using the SYBR Premix Ex TaqTM ll (TaKaRa Biotechnology, Kusatsu, Japan), to quantify the relative expression level of mRNA. Relative mRNA expression was estimated by the double delta delta Ct method and represented as the relative values to the control. GAPDH was used as the housekeeping gene. The primers used for the quantitative PCR are shown in Table 1 . The reaction sensitivity and amplification of the contaminating products were examined by amplifying serial dilutions of cDNA.
7
Quantifying miRNA Expression in Rice Leaves
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Total RNA was extracted from leaves using Trizol reagent (Invitrogen). The RNA quality and quantity were determined by a spectrophotometer (NanoDrop2000 Uvevis). First-strand cDNA was synthesized using the qPCR RT Master Mix with gDNA Remover (TOYOBO). Stem-loop pulse RT-qPCR was performed to analyze the amounts of miR162 with universal reverse primer and speci c forward primer listed in Additional le 4: Table S1 following previous reports (Chen et al. 2005; Varkonyi-Gasic et al. 2007) . U6 snRNA was used as an internal reference to normalize miRNA levels (Turner et al. 2013) . qPCR was performed with speci c primers for the detection of gene expression (Additional le 4: Table S1), and the rice ubiquitin (UBQ) gene was selected as an internal reference gene.
8
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from mammary glands or MAC-T cells using TRIzol (Invitrogen) according to the manufacture's protocol. For quantitative real-time (qRT)-PCR, cDNA were synthesized from total RNA using a qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan).
Quantitative real-time PCR was performed in a Thermal Cycler Dice Real Time System (TaKaRa Bio Inc., Shiga, Japan) by using SYBR Premix Ex TaqTM ll (TaKaRa Bio Inc.). The primer sequences are shown in Table 1. Relative expression was quantified using the standard curve method and data were normalized to β-actin gene (ACTB) expression. All qRT-PCR was performed on Thermal Cycler Dice Real Time System (TaKaRa Bio Inc.), based on a standard curve method. The sensitivity of the reaction and amplification of contaminating products, such as the extension of selfannealed primers, were evaluated by amplifying serial dilutions of cDNA. All data analysis was performed as recommended by the manufacturer.
Quantitative real-time PCR was performed in a Thermal Cycler Dice Real Time System (TaKaRa Bio Inc., Shiga, Japan) by using SYBR Premix Ex TaqTM ll (TaKaRa Bio Inc.). The primer sequences are shown in Table 1. Relative expression was quantified using the standard curve method and data were normalized to β-actin gene (ACTB) expression. All qRT-PCR was performed on Thermal Cycler Dice Real Time System (TaKaRa Bio Inc.), based on a standard curve method. The sensitivity of the reaction and amplification of contaminating products, such as the extension of selfannealed primers, were evaluated by amplifying serial dilutions of cDNA. All data analysis was performed as recommended by the manufacturer.
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